Pyramidal tract neurons drive amplification of excitatory inputs to striatum through cholinergic interneurons

Corticostriatal connectivity is central for many cognitive and motor processes, such as reinforcement or action initiation and invigoration. The cortical input to the striatum arises from two main cortical populations: intratelencephalic (IT) and pyramidal tract (PT) neurons. We report a previously unknown excitatory circuit, supported by a polysynaptic motif from PT neurons to cholinergic interneurons (ChIs) to glutamate-releasing axons, which runs in parallel to the canonical monosynaptic corticostriatal connection. This motif conveys a delayed second phase of excitation to striatal spiny projection neurons, through an acetylcholine-dependent glutamate release mechanism mediated by α4-containing nicotinic receptors, resulting in biphasic corticostriatal signals. These biphasic signals are a hallmark of PT, but not IT, corticostriatal inputs, due to a stronger relative input from PT neurons to ChIs. These results describe a previously unidentified circuit mechanism by which PT activity amplifies excitatory inputs to the striatum, with potential implications for behavior, plasticity, and learning.

(B) Example of EPSCs elicited by the photostimulation of PT fibers while recording from a SPN in ACSF and held at membrane potentials below (-80 mV) and above (-40 mV) the calculated reversal potential for chloride (-75.6 mV). Thin light gray traces are the five individual trials corresponding to the thicker mean traces. (A) From top to bottom: plots showing the normalized amplitude of the 1 st phase of the EPSC, the normalized charge ratio, the normalized peak ratio and the response probability for a 2 nd peak as a function of time, for all SPNs recorded while activating PT inputs in ACSF. Data is mean ± SEM. For each SPN, data was normalized to its first trial. Each time point is the average of 11-62 SPNs from 30 PT-ChR2-EYFP mice.
(B) Example of an individual experiment showing EPSCs from a SPN when identically photostimulating PT fibers in different bath conditions: ACSF (red), MLA (black), and wash out with ACSF (yellow). Thin light gray traces are the five individual trials underlying the thicker mean traces.
(C) From top to bottom: plots showing the amplitude of the 1 st phase of the EPSCs, the charge ratio, the peak ratio and the response probability for a 2 nd peak as a function of time, for the whole experiment in B. Data points are individual trials elicited every 30 s (for charge ratio and peak ratio, circles: EPSC with 2 nd peak; crosses: EPSC without 2 nd peak). Response probability was calculated within a moving window of 3 consecutive trials. Shadowed areas highlight the individual trials (thin light gray traces in B) averaged for each bath condition (red: ACSF; black: MLA; yellow: Wash out). (A) EPSC amplitude of 1 st and 2 nd peak for individual SPNs in ACSF (red) and MLA (black) conditions. n=10 SPNs from 7 PT-ChR2-EYFP mice. Bars represent mean. 1 st peak p=0.69531; 2 nd peak p=0.0039063; Wilcoxon signed-rank test.
(B) MLA modulation index of the amplitude of the 1 st and 2 nd peak for individual SPNs. n=10 SPNs from 7 PT-ChR2-EYFP mice. Bars represent mean. p=0.0039063; Wilcoxon signed-rank test.
(B) Difference in membrane potential between the start and the end of the sag evoked upon a 1 s hyperpolarizing current step of -160 pA from ChIs recorded from IT-or PT-ChR2-EYFP mice. Bars represent mean. IT: n=10 ChIs from 10 mice; PT: n=9 ChIs from 9 mice; p=0.7197; Wilcoxon rank sum test.
(C) Histogram showing the distribution of half spike durations upon a 1 s depolarizing pulse of +60 pA from the ChIs recorded from IT-or PT-ChR2-EYFP mice. IT: n=74 spikes from 10 ChIs from 10 mice. PT: n=65 spikes from 9 ChIs from 9 mice; p=0.3982; Wilcoxon rank sum test, z=0.84.
(D) Histogram showing the distribution of inter-spike intervals upon a 1 s depolarizing pulse of +60 pA from the ChIs recorded from IT-or PT-ChR2-EYFP mice. IT: n=64 intervals from 10 ChIs from 10 mice. PT: n=56 intervals from 9 ChIs from 9 mice; p=0.18065; Wilcoxon rank sum test, z=1.34.

Fig. S8. 4-AP and TTX abolish the differences between IT-and PTSPN EPSCs
(A) Probability of evoking a 2 nd peak as a function of the 1 st peak amplitude when photostimulating IT (blue) or PT (red) axons. Data is binned in 5 bins of 30 pA. The number of trials with 2 nd peak/total number of trials for each bin is: IT, 7/23, 14/35, 7/35, 4/22, 6/14 from 10 SPNs from 10 IT-ChR2-EYFP mice; PT, 9/90, 5/47, 4/10, 1/4, 0/1 from 10 SPNs from 9 PT-ChR2-EYFP mice. See Figure 1I for comparison. Note that the biphasic events in these experiments are likely to be similarly overestimated for IT-and PTSPN EPSCs due to the intrinsic occurrence of multiphasic events in these pharmacological conditions (49).